ain aur,TnaMri,WnSiadJreFilmus* Jorge and Shi Wen the Martin, Tonya in Capurro, Mariana role signaling direct Wnt a canonical plays of and stimulation Frizzled to binds -3 ARTICLE RESEARCH ß eevd1 uut21;Acpe aur 2014 January 6 Accepted 2013; August 16 Received ([email protected]) correspondence for *Author 3M5, M4N ON Toronto, Avenue, Bayview Medical 2075 of Toronto, Department of and University Institute, Biophysics, Research Sunnybrook Sciences, Biological including pathways, signaling several regulate can glypicans 2008). that al., et suggesting (Filmus surface, membrane the cell cell places other the with is This interact to 1999). could family close al., chains et are this Another GAG (Veugelers sites These C-terminus the of 1997). chains. the GAG to members the al., close for located the sites display et insertion by the also of (Saunders localization shared can sulfate carry feature (GPC5) chondroitin structural glypicans glypican-5 of but general, chains chains, In linear sulfate long being chains. five heparan to after two (GAG) display environment to al., glycosaminoglycan et shown extracellular Traister been 2004; have al., the et Glypicans (Kreuger 2008). Notum in called lipase addition, found a In by released 2012). be Capurro, the can and Filmus glycosylphosphatidylinositol they of 2008; a al., by outer et members the membrane (Filmus anchor mammalian to plasma bound the six are of Glypicans surface of proteoglycans. of one family glypican is (GPC3) Glypican-3 INTRODUCTION Frizzled sulphate, Heparan , Proteoglycan, Wnt, Glypican-3, membrane. WORDS: that cell KEY the finding at the Wnt to fact by binds the provided despite it signaling, that is Wnt canonical model inhibits (GPC6) our glypican-6 GPC3. for triggers and membrane Frizzled support Wnt, cell includes the Additional that at complex with Wnt a of of Consistent endocytosis binding the of the Frizzled. that formation and show the we Wnt this, stimulates between glypican complexes this signaling that indicating chains GPC3, glycosaminoglycan the of GPC3 signaling. through Wnt, Wnt directly with interact of interacting that Frizzled to stimulation and addition the demonstrate in in that, we show role we direct study, Specifically, more this a plays in signaling membrane, GPC3 its However, cell with factor the Frizzled. growth stimulate at this , of proteoglycans Wnt interaction of the these amount facilitating that thus the proposed increasing by been signaling has by growth with it interact cell HCC glypicans Wnts, stimulates Because the signaling. GPC3 Wnt to hepatocytes. canonical promoting bound normal (HCCs) by carcinomas is hepatocellular not most that but by proteoglycan expressed is a It surface. is (GPC3) Glypican-3 ABSTRACT 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,16–55doi:10.1242/jcs.140871 1565–1575 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. eei n iceia tde aedmntae that demonstrated have studies biochemical and Genetic ebre n asn 03 u ta. 03,bone 2003), al., et (FGFs) factors growth 2008; fibroblast and (Gutie Lum 2003) al., al., et Dwivedi Fujise 2004; 2013; et 2003; al., al., et et (Capurro (Belenkaya Sanson, (BMPs) (Hhs) proteins morphogenetic Hedgehogs and 1999), Desbordes al., 2001; al., et et Topczewski 2005; Tsuda al., et (Song Wnts by triggered those ntepooino elpoieainadsria Cees 2006; (Clevers, survival involved and are proliferation which cell of of nucleus, promotion some the , the in many factor the In of expression nucleus. induces the transcription the drives binding to the migrates Wnt subsequently of 2012). 6, or (Niehrs, 2000; accumulation 5 receptor-related LRP5/6) al., the lipoprotein and et hereafter (low-density Frizzled, co-receptors: LRP6 with Satoh two or to Wnt 2002; LRP5 of associated binding normally al., is the pathway by events signaling triggered et This 2007). (Kern Monga, molecular and HCC Thompson frequent of progression most the 2013). this al., regulate et Kamimura to 2010; shown al., et been (Buraschi have pathway signaling be proteoglycans should it other addition, In that 2009). al., noted et Yan 2012; al., et Sakane 2012; oei euaigrcpo–iaditrcin,gyiasalso glypicans interactions, receptor– their regulating glypicans a to addition in of in In present function. features are glypican role determine that structural to molecules type signaling the cell or of given that set stimulatory the is with a combine studies either emerging is these that have picture from The can activity. signaling glypicans on effect inhibitory and ligand the receptor, between interaction the and of level the at occurs signaling of oso-ucinsuishv eosrtdta te mammalian in other and that signaling demonstrated Wnt have been and Gain-of-function studies regulates 2005). loss-of-function al., has et mice GPC3 (Song GPC3-null finding tissues that embryonic of normal Studies This demonstrated 2012). also 2005). al., et have GPC3 al., (Li HCC, others et by of canonicalconfirmed (Capurro marker promoting by signaling a malignancy being this Wnt of to growth addition has the in laboratory stimulates our that, from shown Work Marrero, also 2010). 2011; Kojiro, Sherman, and and to Roskams (Bruix 2009; pathologists HCC clinical of by diagnosis used a being confirm currently immunohistochemical is GPC3 the of Consequently, detection 2005). al., 2006; al., et et Libbrecht Yamauchi 2003; al., hepatocytes et (Capurro normal lesions by benign and not but (HCCs) carcinomas hepatocellular 2009). al., et (Capurro pathway signaling Hh is the activity regulatory by this mediated that showed (Capurro we Furthermore, bone 2009). developing al., the et in proliferation cell We of 1998). regulation the in al., role important et an plays (Pellegrini glypican this tissues that previously demonstrated adult 2010). most al., in et downregulated Wu 2004; al., et Han 2008; the al., in et gradients Gallet BMP and Hh Wnt, Drosophila of formation the in participate ciaino h aoia n inln aha soeof one is pathway signaling Wnt canonical the of Activation ti o eletbihdta P3i xrse ymost by expressed is GPC3 that established well now is It is it but development, during expressed widely is GPC3 Drosophila ´ rzadBadn 00.Tegyia-eitdregulation glypican-mediated The 2010). Brandan, and rrez mgnlds Aiaae l,20;Aese l,2010; al., et Ayers 2008; al., et (Akiyama disc imaginal lpcn lorglt n inln Fc tal., et (Fico signaling Wnt regulate also glypicans b ctnn which -catenin, b -catenin 1565

Journal of Cell Science hyd o aeactpamcdmi htcudtransduce could that domain that and cytoplasmic 2009), a al., have et Yan not 2012; do Wnt–Frizzled al., they et (Sakane specific membrane 2005). cell only al., pathway et canonical Takada type, the 2010; al., activate et cell to human (Grumolato that, capacity the and the the genes, have that Frizzled interactions noted 10 on and be genes should depending Wnt It 19 includes 2006). genome Nusse, and Gordon ARTICLE RESEARCH 1566 a the by which driven in transiently was vector were reporter cells luciferase a a a 293T and of as end, ligand– GPC3 expression GPC3 on this with GPC3 of To of transfected absence impact affinity. the and Wnt3a assess receptor increasing to presence experiment of we the surrogate activity assumption, signaling in this the on concentrations measure Based with to occupancy. correlates decided concentrations receptor Wnt a overall in increasing observed the at activity type signaling the cell the Alternatively, that given interpret. assume proteins, to to reasonable difficult Wnt-binding is be the it would other plots and such and of express Wnt results proteins cells because between Frizzled However, cells. interaction several intact Scatchard on the generate directly complex to to receptor To necessary cell. corresponding be target would plots the it or the by hypothesis, Frizzled expressed having relevant this the test are GPC3 for that Wnt on cell receptors of based affinity LRP the the be increase of to could ability signaling level Wnt the canonical at acting activation. LRP6 by of stimulates upstream GPC3 signaling membrane, that result Wnt hypothesis This cells the canonical control. for GPC3-transfected vector support with the strong provides transfected in cells in tested with increase compared were significant Wnt the that a all anti-phospho- were at 1A, concentrations observed an Fig. was levels in medium with phospho-LRP6 shown Wnt3a-induced analysis conditioned As blot antibody. western control LRP6 using by or of assessed levels medium Wnt3a- the of and dilutions with different conditioned vector, cells by induced control 293T were the with transfected that at phospho-LRP6 or We receptors GPC3 2007). signaling encoding al., its vector et to (Bilic Wnt events surface of earliest well cell binding the can is of the one by GPC3 It is triggered whether phosphorylation LRP6. this investigated of directly that established to phosphorylation first Here, Wnt3a-induced we 2008). stimulate al., hypothesis, et this we (Filmus 2005), test reception the al., at signal activity et of Wnt Song canonical level stimulates 2005; GPC3 interact al., that can et proposed GPC3 have (Capurro that luciferase finding Wnts our the a various on with induces and using stimulates results, GPC3 these by on ectopic Based GPC3 that cytoplasmic cells showing of that HCC by accumulation and in assay, previously activity reporter demonstrated Wnt canonical have reception signal We of level the at activity Wnt3a stimulates GPC3 RESULTS Frizzled. on with based glypican signaling, this Wnt of canonical role of interaction direct stimulation the the more in a Here, GPC3 supporting 2012). of al., evidence et experimental (Sakane provide receptors we signaling in the Wnt of of proteoglycans concentration vicinity these the the increasing that by proposed signaling been Wnt stimulate has it activity, signaling ei,adtelcfrs ciiywsmaue.A hw in shown As measured. was activity luciferase control-conditioned the or and Wnt3a-conditioned media, with of incubated then dilutions were different cells Transfected promoter. responsive ae ntefc htgyiascnitrc ihWt tthe at Wnts with interact can glypicans that fact the on Based n osbemcaimfrteGC-nue tmlto of stimulation GPC3-induced the for mechanism possible One b ctnn(aur ta. 2005). al., et (Capurro -catenin b -catenin- rnfce 9Tclswt xrsinvcosfrGC and GPC3 for transiently vectors we expression We Frizzled, with to cells and approach studies. first 293T GPC3 a transfected co-immunoprecipitation between As Frizzled. interaction performed with to this interacting the bind test to also investigate addition To can in GPC3 signaling. whether, Wnts, Wnt studied of we hypothesis, stimulation in involved the is GPC3-induced mechanism and a similar the we a receptor knowledge, of whether this investigate FGF on to assembly Based the decided 2004). al., the FGF, et (Ibrahimi includes requires proteoglycan that proteoglycans complex of which stimulation by ternary the at that signaling level established well the is FGF at It received? signaling is Wnt signal the stimulate GPC3 could How Frizzled with interacts GPC3 eit h neato Fg B.Fnly einvestigated we Finally, 2B). Frizzled (Fig. the non- interaction chains of the sulfate any heparan the of the with that binding mediate covered indicating significant observed, beads was No proteins to mutated 1998). been GPC3 have al., glycanated chains sulfate et G heparan (Gonzalez the experiment for Frizzled-covered pull-down sites the insertion the a of GPC3 incubating end, with beads role by this performed To the the was characterize chains. investigated further sulfate indicating To we heparan shown), FZD7. interaction, not to (data directly in GPC3–Frizzled step binds observed the acid-wash that GPC3 to the that lysing similar of was was absence before that FZD7 GPC3–AP the be acid-washed of step amount could the the to that that acid-wash bound found proteins We endogenous an FZD7. to remove bound of to cells, assay HCC addition pull-down transfected that GPC3– the in the found the repeated that we we expressed confirm with 2B, direct, To Fig. was also GPC3. interaction to in bind Frizzled are shown FZD8 and As which FZD7 2008). both FZD8, al., et We and (Bengochea FZD4. interact can FZD7 with GPC3 FZD4, interacts to with addition GPC3 in 2B, that whether, investigated Fig. indicating also than in alone, more shown significantly AP As beads that does measured. FZD4-covered activity was to AP binds beads of GPC3–AP amount the and the by washing, retained antibody After was alone. AP anti-FLAG then protein with fusion were or (AP) beads an the GPC3–alkaline-phosphatase The a and beads. with with incubated G lysed, Protein expression were incubated with FZD4 cells immunoprecipitated a were transfection, with after lysates transfected days were Two cells interaction vector. To the 293T assays. the pull-down end, study performed in to this we co-immunoprecipitates Frizzled, approach FZD4 and alternative FZD4 GPC3 was that an between found As As of analysis. GPC3 we GPC3. blot 2A, with Fig. western lysed, presence by in assessed shown 2008). were the was al., is material cells et precipitated and it Bengochea the 2008; and immunoprecipitated al., transfection, cells, et (Pan 293T Following HCC in in signaling expressed mediates canonical protein Frizzled Wnt3a-induced This (FZD4). Frizzled-4 FLAG-tagged n3 ipasahge idn fiiyfrFize nthe in Frizzled for affinity binding glypican. this that higher of idea presence the a the with in consistent displays obtained this were were Wnt3a GPC3 the although that of absence curves affinities, that, and activity binding presence noted luciferase measure the be to of also designed slopes between not should interaction the was It signal- of experiment Frizzled. a level the for and at support Wnt GPC3 Wnt3a- additional of role Wnt3a. provides increases stimulatory of result significantly concentrations tested this GPC3 all Therefore, at that activity luciferase found induced we 1B, Fig. ora fCl cec 21)17 5517 doi:10.1242/jcs.140871 1565–1575 127, (2014) Science Cell of Journal D A–P uinpoeni hc h GPC3 the which in protein fusion a GAG–AP,

Journal of Cell Science EERHARTICLE RESEARCH hs rtiswt h eaa uft hiso GPC3. of results chains sulfate These heparan 2D). the with (Fig. proteins of CRDs these interaction the mediate Frizzled proteins Frizzled of the CRDs the that indicate of GPC3 any non-glycanated with interact the CRDs full- the Frizzled Frizzled, with the observed results all that length the to found with Consistent or GPC3–AP we tested. were 2D, of FZD7 that Fig. binding FZD4, in substantial shown with is As there covered GPC3–AP. beads with CRDs pull- incubating further performed FZD8 we by end, to this assays To Thus, GPC3. down to Wnts. bind can with CRD Frizzled whether interact the investigated domain we to interaction, cysteine-rich Frizzled–GPC3 known the a characterize N-terminal is contains The which cell. GPC3, Frizzled (CRD), the with of of interact portion outside cannot extracellular completely therefore located proteins is these which provides of part interaction. result GPC3–Frizzled large role the in essential This chains an sulfate heparan 2C). supporting the (Fig. for evidence manner experimental to in additional binding dose-dependent GPC3–AP this inhibits of heparin binding expected, a as the that, found with We compete FZD7. can heparin whether (Ser phospho-LRP6 of levels the (W3A)-conditioned and Wnt3a lysed of then amounts were indicated the Cells with h. stimulated 1 were cells for Transfected level. medium (EF). signal-reception (L)-conditioned control vector the control or at GPC3 activity with transfected Wnt3a transiently stimulates GPC3 1. Fig. P3o etrcnrl(E)(2 g.A 6hatrtaseto,clswr rpiie,rpae no2-elpae 4,0 el/el n incub and cells/well) (40,000 eff transfection plates for mean 24-well normalized the onto measured, represent then replated was data trypsinized, activity the were Luciferase and h. cells a Wnt3a quadruplicate, 2.5 ng), transfection, various for (500 after control at reporter h medium activity luciferase L 16 Wnt3a Wnt-responsive or the At in Wnt3a-induc medium a stimulates bottom ng). Wnt3a-conditioned of with GPC3 of the transfected (125 level (B) dilutions at were The (pEF) various results. Numbers and calculated. control 1. similar plate then vector of 6-well with was or value a times LRP6 in a GPC3 three seeded total assigned repeated were to arbitrarily cells was was P-LRP6 293T dose experiment of concentrations. Wnt3a This ratio lowest ratios. The the software. P-LRP6:LRP-6 with ImageJ relative cells NIH vector-control-transfected using of quantified phosphorylation and LRP-6 scanned were Bands blotting. rzldpoen r ee-pntasebaercpos n a and receptors, transmembrane seven-span are proteins Frizzled b glcoiaeatvt,adtertobtentelcfrs ciiyi h rsneadasneo n3 a acltd ahsml a performe was sample Each calculated. was Wnt3a of absence and presence the in activity luciferase the between ratio the and activity, -galactosidase 6 ..Teeprmn a efre wc ihsmlrresults. similar with twice performed was experiment The s.d. D A–Pddnot did GAG–AP A P3siuae n3-nue hshrlto fLP.23 el were cells 293T LRP6. of phosphorylation Wnt3a-induced stimulates GPC3 (A) ofclmcocp.A hw nFg ,w on ht as that, found displayed FZD8–YFP we and 3, GPC3 Fig. expressed in that shown cells a using expected, As by observed microscope. were then and confocal were Wnt, cells and GPC3 The for fixation. immunostained before proceed to endocytosis allow ahdadfxd rwr rnfre o37 to transferred were or fixed, and washed odtoe eimfr1hu t8 at hour Wnt3a-containing 1 with for incubated medium were conditioned cells day One protein. transfection, fusion with FZD8–YFP after cells a 293T and that GPC3 co-transfected hypothesize encoding complex. we vectors to molecular possibility, internalized reasonable this the is investigate in To present it be case, also the will a GPC3 is both form proteins with this three interacts If these is that GPC3 Yamamoto complex. suggest that 2006; strongly showing Frizzled there Nusse, and results Wnt and Our controversial, 2008). (Blitzer al., endocytosis signaling et still Wnt-induced for essential that is is suggesting endocytosis evidence the convincing Although of 2006). Wnt– Nusse, the mechanism and of cell-surface (Blitzer its endocytosis complex to induces Wnt3a Frizzled–LRP LRP5/6 of and binding Frizzled the that receptors Wnt3a established and well Frizzled is with It along endocytosed is GPC3 ora fCl cec 21)17 5517 doi:10.1242/jcs.140871 1565–1575 127, (2014) Science Cell of Journal 1490 PLP)o oa R6wr sesdb western by assessed were LRP6 total or (P-LRP6) ) ˚ .Teclswr then were cells The C. b glcoiaevco and vector -galactosidase ˚ o 5mntsto minutes 75 for C cec using iciency eimo L or medium tdwith ated iaethe dicate 1567 din ed

Journal of Cell Science EERHARTICLE RESEARCH 1568 GPC3–FZD8, is of route internalization endocytic (Sakane Wnt3a-induced same route the the with whether during caveolin-mediated investigate along used To a Wnt3a, 2012). through al., to et LRP6, response and in FZD2 internalized is (glypican-4) and proceed. Wnt3a to internalized allowed is with is complex endocytosis this complex that when and a surface, cell forms the at GPC3 FZD8–YFP similar that in suggests incubated result strongly were This (inset). cells medium control-conditioned with when but conditions occur 3C). not (Fig. cytoplasm did the Endocytosis in observed were 3B). Wnt3a and (Fig. colocalized FZD8 with GPC3, vesicles surface staining of number membrane cell large a the and the disappeared, mainly endocytosis, at Wnt3a-induced bound upon Wnt3a Notably, of levels detectable Frizzled. with interacts GPC3 2. Fig. oto ed.Teeprmn a efre he ie ihsmlrrsls ih ae,wsenbo nlsscnimn httebasdslys display beads the that right. confirming the analysis on blot indicated western are panel, markers Right activ molecular-mass AP results. of the similar positions of G with subtraction The Protein after times Frizzled-CRDs. and beads three Frizzled-CRD-covered bound antibodies the performed of anti-Myc to was with amounts bound experiment triplicates) incubated Results of The and (mean+s.d. heparin. beads. lysed activity hep of control were AP of concentrations specific cells GPC3 Effect the GPC3–AP, indicated the represent (C) with later, the bars incubated right. days of panel, then Two the presence were control. on the beads vector indicated The in or are beads. above FZD8-CRD markers described results FZD7-CRD, molecular-mass as similar FZD4-CRD, of performed with Myc-tagged positions was times The assay four mean beads. pull-down bou performed the the triplicates) A was represent to of interaction. experiment (mean+s.d. attached GPC3–FZD7 The activity were the beads. AP proteins on control specific Frizzled GPC3 the the GPC3–AP, of represent to with cont amounts Bars incubated bound vector similar measured. then activity or Student’s was were AP FZD8 beads unpaired beads the FZD7, the The an beads. by FZD4, of experiments, posi G retained FLAG-tagged subtraction Protein The activity with after and AP control. transfected beads antibodies the vector were (lower anti-FLAG Frizzled-covered EF, washing, with cells GPC3 After A; 293T incubated immunoprecipitated alone. hemagglutinin and assays. the AP and lysed HA, Pull-down in or panel) were chain; (B) FZD4 cells (middle light left. of days, lysates immunoglobulin the presence 2 whole-cell on LC, After the in indicated immunoprecipitation; panel, FZD4 IP, are Upper of analysis. markers antibody. Levels blot molecular-mass anti-HA (WB). western an blotting by using western assessed by by were immunoprecipitated assessed was as GPC3 material and precipitated vectors, expression control corresponding shsbe eotdrcnl htaohrmmainglypican mammalian another that recently reported been has Is 6 ..o rpiae.Teeprmn a efre he ie ihsmlrrsls D uldw sas 9Tclswr rnfce with transfected were cells 293T assays. Pull-down (D) results. similar with times three performed was experiment The triplicates. of s.d. t ts eeldahgl infcn P3Fize idn ( binding GPC3–Frizzled significant highly a revealed -test A oimnpeiiain 9Tclswr rnfce ihH-agdGC,FA-agdFD (FZD FZD4 FLAG-tagged GPC3, HA-tagged with transfected were cells 293T Co-immunoprecipitation. (A) D A–Po Paoe fe ahn,teA ciiyrtie ytebaswsmaue.Left measured. was beads the by retained activity AP the washing, After alone. AP or GAG–AP noyoi Fg F.Ti eutidctsta P3is a GPC3 through part, that in least indicates at FZD8, result route. and caveolin-mediated Wnt3a This with Wnt3a-induced during internalized 3F). (Fig. formed are not a in that endocytosis membrane, do occurs vesicles caveolin cell FZD8 cytoplasmic and the and FZD8 the at GPC3, GPC3 are of they whereas colocalization significant when that caveolin found additional with shown we colocalize the As 3D,E, 3D–F). with Fig. (Fig. in repeated caveolin endogenous was of immunostaining experiment endocytosis the hl ewr efrigsuiso h ucino another of function a (GPC6), the glypican-6 family, on glypican mammalian studies the of performing member were Wnt3a we inhibits but level While surface, signal-reception cell the the at at activity Wnt3a to binds GPC6 ora fCl cec 21)17 5517 doi:10.1242/jcs.140871 1565–1575 127, (2014) Science Cell of Journal P , .0) oe ae,wsenbo nlsscnimn that confirming analysis blot western panel, Lower 0.001). t on othe to bound ity 4 D rthe or ) GAG–AP in of tions dt the to nd nall In . imilar arin panel) rol.

Journal of Cell Science ahdwieln oe akeape fedctssrgos(yolsi eils CF.Pasnscreaincefcet o h niae p indicated the boundarie for cell coefficients mark correlation lines Pearson’s white (C,F). Dotted vesicles) medium. (cytoplasmic conditioned regions (mean CM, endocytosis right colocalization. of the indicates examples on picture mark included merged boxes the line in white white dashed or Yellow immunostained. then EERHARTICLE RESEARCH n3 osntbn oanngyaae P6(GPC6 GPC6 non-glycanated of a capacity to that Wnt3a-binding bind observed we not the Interestingly, does GPC3. that of beads Wnt3a that than found control higher to is also to GPC6 binds We than Wnt3a that more 5B). (Fig. observed significantly We transiently with beads assay. interacts pull-down GPC6-covered a cells GPC6 a As whether in Wnt3a–HA. co- investigated 293T Wnt3a and we Wnt3a GPC6 approach, in that encoding second found vectors GPC6 we with co-immunoprecipitaion transfected 5A, performed with Fig. first in immunoprecipitates we shown As end, experiments. this To Wnt. reduced GPC6 4B). Wnt because (Fig. reception, phosphorylation signal inhibition canonical LRP6 of This Wnt3a-induced level 4A). inhibited the (Fig. at manner significantly occurred dose-dependent a glypican in to signaling contrast in this that, revealed cells GPC3, 293T in assay reporter luciferase 8 at h 1 for (Wnt3a) medium Wnt3a-conditioned complexes. or (L) GPC3–FZD8–Wnt3a medium of control-conditioned endocytosis with the incubated induces Wnt3a 3. Fig. eemn hte,lk P3 P6bnst n3 ttecell the definitively at Wnt3a To chains, to binds sulfate sites. GPC6 heparan GPC3, Wnt3a-binding of like the interaction whether, multiple by the determine GPC3, mediated display unlike is that, the which Wnt3a fact that the that with to likely with GPC6 due compared highly is as is GPC3 GPC6 of It of capacity 5C). Wnt3a-binding (Fig. greater assay observed reporter we luciferase finding, this with GPC6 GPC6–Wnt3a that Consistent the 5B). for required (Fig. are interaction chains GAG the that indicating 37 ˚ eteeoedcddt netgt hte P6bnsto binds GPC6 whether investigate to decided therefore We o 5o 0mnt lo noyoi opoed(,,) P3(le n n3 rd AC,o P3(le n aeln1(a-)(e)(–)were (D–F) (red) (Cav-1) caveolin-1 and (blue) GPC3 or (A–C), (red) Wnt3a and (blue) GPC3 (C,E,F). proceed to endocytosis allow to min 80 or 75 for C D A sual oihbtcnnclWtsgaigi a in signaling Wnt canonical inhibit to unable is GAG 6 ..o tlat1 ersnaieiae) cl as 10 bars: Scale images). representative 14 least at of s.d. D GAG), 9Tclswr rnfce ihGC n Z8YP(Z) n were and (FZD), FZD8–YFP and GPC3 with transfected were cells 293T muto n3 ttecl ebaei o nuhto enough not in membrane the cell increasing the that at signal-reception indicate signaling. Wnt3a the stimulate observations to of at contrast these activity amount in Thus, However, Wnt3a membrane. inhibits level. cell GPC6 the at GPC3, Wnt3a cell to the binds at Wnt3a to chains. GAG binds its GPC6 through GPC6 that surface or therefore, control was vector conclude, binding with to We no transfected whereas bound cells 5D), only in (Fig. Wnt3a detected GPC6 expressed that that assessed found cells was We the cells the immunostaining. to using Wnt3a by of binding the material, unbound i.6,FD osntc-muorcptt ihGPC6, this with confirm To co-immunoprecipitate interact. not do not proteins in two shown does these that As indicating co-immunoprecipitation FZD4 cells. performed 293T 6A, approach transfected Fig. we first transiently hypothesis, a in As this experiments Frizzled. and test Wnt to with complex form cannot signaling and Frizzled a with interact not does GPC3, to contrast GPC6, Wnt in that inhibit is but possibility surface, One level? cell signal-reception the the at at signaling Wnt3a to bind GPC6 Frizzled could with How interact not does GPC6 lcntdGC rvco oto,adicbtdte with them incubated and 8 non- control, at GPC6, vector medium with Wnt3a-conditioned or cells GPC6 293T transfected glycanated transiently we surface, ae oehr hs eut hwta,lk P3 GPC6 GPC3, like that, show results these together, Taken m m. ora fCl cec 21)17 5517 doi:10.1242/jcs.140871 1565–1575 127, (2014) Science Cell of Journal ˚ .Teclswr hnete ie ABD rtaserdto transferred or (A,B,D) fixed either then were cells The C. ˚ .Atrwsigt eoethe remove to washing After C. rnr are artners D ,and s, GAG. 1569

Journal of Cell Science EERHARTICLE RESEARCH 1570 8 control, a at as hours AP 2 or vector- GPC6–AP for either or of activities FZD4- equal medium end, containing conditioned with this incubated were To cells by control-transfected assay. cells cell-binding intact a in assessed performing was interaction GPC6–FZD4 valu the a result, assigned of arbitrarily levels were the LRP cells * and phosphorylated vector-control-transfected of experiments. lysed, wa of ratio then independent experiment The phosphorylation This software. were three LRP-6 ImageJ (LRP6). Cells of Wnt3a-induced NIH levels h. mean+s.d. using of LRP6 quantified vector 1 the levels total and expression for The represent for scanned GPC6 calculated. medium were re-probed bands or then then (L)-conditioned panel, was GPC3 was control Lower molecular-mass membrane with LRP6 results. or The of similar transfected (W3a)- blotting. with positions were Wnt3a times western The cells three by with 293T proteins. repeated assessed stimulated LRP6. core were were of full-len non-glycanated (P-LRP6) and phosphorylation the (Ser1490) immature pCDNA), Wnt3a-induced phospho-LRP6 to the (d or inhibits (directed of 1G12 (EF GPC6 antibody antibody detection (B) control anti-GPC3 polyclonal the the right. vector anti-GPC6 by indicate antibody the detected an Arrows anti-HA on is by the C-terminus). GPC3 corresponding indicated detected glycanated the by the are to and is detected in corresponding GPC6 GPC6 smear located bands or a glycanated GPC3 epitope non-specific panel, HA-tagged to site an indicate Right with corresponding conditions). cleavage (*) transfected blot smear internal cells asterisks western 293T a an The non-reducing of protein, panel, contain analysis detected. blot Left glypicans is Western pCDNA). Both tag cells. (EF, 293T assay. HA in controls luciferase the glycanated the are with glypicans for subunit both transfected N-terminal panel, were the that only Th ( cells and the subunit triplicates). separate, of (N-ter) in levels (mean+s.d. N-terminal GPC3 an Wnt3a and by generates ( induced GPC6 Student’s luciferase tested assess stimulation unpaired a doses to fold The with all antibody, the results. along at represent similar EF), respectively, Bars with or GPC3, performed. times (pCDNA and was four alone assay repeated vector luciferase was and or a (TOPFLASH) experiment vectors, promoter and expression responsive lysed GPC3 Wnt medium, HA-tagged canonical conditioned a or by GPC6 level. driven HA-tagged signal-reception vector of the reporter amounts at increasing activity with Wnt3a transfected inhibits GPC6 4. Fig. oiiecnrl ssoni i.6,w i o e any see FZD8, not and FZD7 We with a did cells. interact intact can cells, GPC6 in as we whether interact FZD4-expressing investigated not also do the used 6B, proteins Cells Fig. two to these were that GPC6–AP measured. in indicating of protein shown was binding fusion significant As remained GPC3–AP control. that a positive activity with AP incubated of amount ˚ .Clswr hnwse n ye,adthe and lysed, and washed then were Cells C. , 0t 0ka n -emnlsbnt ern h A his ne euigcniin,tetosubunits two the conditions, reducing Under chains. GAG the bearing subunit, C-terminal a and kDa) 40 to 30 P , .0) idepnl etr ltaayi htwspromdi euigcniin iha anti-HA an with conditions reducing in performed was that analysis blot western panel, Middle 0.001). P 5 0.005. A pe ae,GC niisWtaidcdlcfrs ciiy 9Tclswere cells 293T activity. luciferase Wnt3a-induced inhibits GPC6 panel, Upper (A) t b ts eeldhgl infcn niioyadsiuaoyefcso GPC6 of effects stimulatory and inhibitory significant highly revealed -test swssonfrGC.W on htnn fteeFrizzled these of none that 6B). found (Fig. GPC6–AP We to GPC3. bound for proteins shown was as ihorfnigta P3i nenlzdi opee htalso consistent that complexes is in This internalized membrane. is GPC3 cell that the finding complex at our a with Frizzled form can and GPC3 results Wnt that other propose with Wnt, on we and with paper, this, molecular this interacting on Wnt in Based the included to Frizzled. canonical to addition into binds in glypican of insight this that, showing stimulation crucial by GPC3-induced provide signaling, of we study, mechanisms this In DISCUSSION glcoiae el eete tmltdoengtwt n3-o control- or Wnt3a- with overnight stimulated then were Cells -galactosidase. ora fCl cec 21)17 5517 doi:10.1242/jcs.140871 1565–1575 127, (2014) Science Cell of Journal f1 Bars 1. of e rce to irected ototal to 6 markers Lower . e vector gth that sor s

Journal of Cell Science EERHARTICLE RESEARCH noyoi fteGC–n–rzldcmlx However, complex. GPC3–Wnt–Frizzled signaling-productive the of of levels endocytosis receptor. the the thus and in ligand interaction, increase the Wnt–Frizzled between an the complexes of presence of to the affinity that leading the propose also raises We GPC3 FZD8. and Wnt3a contain nonspec (*), Asterisks cells. weste and transfected panel, TOPFLASH the GPC6 right with of (C) Lower along lysates right. detected. vectors the ( the is expression in on experiments beads indicated GPC3 shown independent control the and glypican- are three pCDNA with GPC6 indicated markers of and transfected of the Molecular-mass mean+s.d. EF were levels to antibody. the to cells the bound anti-HA 293T as Wnt3a assessing Wnt3a the shown of antibody panel) by of are binding (upper anti-HA amount detected Data blotting non-specific an bands the western beads). Some with by represent control significant). assessed performed bars to was not analysis panel, beads bound NS, blot the left Wnt3a bars; to Lower mediu of the bound software. (W3a)-conditioned amount was above Wnt3a NIH that the indicated of Wnt3a ImageJ to of amounts with (relative amount equal were analyzed the beads with that material, and incubated covered cells unbound scanned then 293T the were were remove of beads to blots Lysates was The washing assay. The After lysates beads. Pull-down h. G whole (B) cha 2 Protein in right. for light and levels (CM) the antibody immunoglobulin GPC6 anti-HA on LC, of an shown immunoprecipitation; GPC3 assessment using IP, lysa are GPC3, The immunoprecipitated whole-cell GPC6. markers HA-tagged in blotting. glycanated Molecular-mass with panel) western the A. w (second transfected by of material hemagglutinin Wnt3a transiently assessed detection and HA, precipitated were panel) the the chain; panel) (first allow heavy in GPC6 (fourth which Wnt3a immunoglobulin of material conditions, of levels precipitated non-reducing The presence the under panel). The in (third performed immunoprecipitated. antibody GPC6 was anti-HA of chains. GPC6 an presence GAG and with the the (WB) vectors, through blotting expression western surface control by cell corresponding assessed the the at or Wnt3a Wnt3a with tagged interacts GPC6 5. Fig. h -emnlsbntpoietebs seseto xrsinlvl.()GC id oWtaa h elsrae 9Tclsta eetransfected were that cells 293T surface. cell the at t Wnt3a conditions to reducing in binds under levels GPC6 that expression (D) Note Wnt3 GPC6 levels. bands. by of expression nonspecific induced analysis of (*), stimulation blot assessment Asterisks fold GPC6 western best right. the or panel, the the represent GPC6 Lower provide on bars results. subunit indicated panel, similar N-terminal are Upper with the markers performed. twice Molecular-mass was repeated cells. assay was 293T luciferase experiment transfected a The and triplicates). lysed of medium, (mean+s.d. control-conditioned L or Wnt3a- muotie.Ylo nmre itrsidctsclclzto.Saebr:10 bars: Scale colocalization. indicates pictures merged in Yellow immunostained. loson(mean shown also nti td,w aentasse h oeo R56i the in LRP5/6 of role the assessed not have we study, this In D A eeicbtdwt oto L rWtacniindmdu o t8 at h 1 for medium Wnt3a-conditioned or (L) control with incubated were GAG 6 ..o 5rpeettv images). representative 15 of s.d. D A,GC rGPC6 or GPC6 GAG, D A xrsinvcoso h orsodn oto etr were vectors control corresponding the or vectors expression GAG m ewe P3advrosFrizzled various and GPC3 the between Frizzled of and part be ligand also this would LRP5/6 canonical complex. with that of GPC3-containing expect complex presence we the a 2012), in (Niehrs, forms that, LRP5/6 established well Wnt, is it because .Pasnscreaincefcetfrclclzto fGC n n3 is Wnt3a and GPC6 of colocalization for coefficient correlation Pearson’s m. A oimnpeiiain 9Tclswr rnfce ihGC,HA- GPC6, with transfected were cells 293T Co-immunoprecipitation. (A) eue w ifrn prahst eosrt h interaction the demonstrate to approaches different two used We ora fCl cec 21)17 5517 doi:10.1242/jcs.140871 1565–1575 127, (2014) Science Cell of Journal b glcoiae h el eete tmltdoengtwith overnight stimulated then were cells The -galactosidase. ˚ n ie.GC gen n n3 rd eethen were (red) Wnt3a and (green) GPC6 fixed. and C D A osntihbtWtaidcdlcfrs activity. luciferase Wnt3a-induced inhibit not does GAG rtis co-immunoprecipitation proteins: P elvl of levels he vle are -values n HC, in; the e,and tes, 1571 ific with a rn as m .

Journal of Cell Science EERHARTICLE RESEARCH elsrae u bevto ht nieGC,GC osnot does 1572 GPC6 an GPC3, the as unlike at that, Wnt3a acts observation bind the can Our GPC6 glypican surface. this increase signaling, cell that by fact Wnt to the here which despite in provided canonical inhibitor, context ability cellular is stimulates same surface the its in GPC3 cell that, show beyond the which results, at our goes ligand protein of signaling same concentration the Wnt of in part are LRP6 and in complex. FZD2 model vesicles a GPC4, endocytic with in consistent which and is membrane internalization cell with Wnt3a-induced the colocalizes after on GPC4 that LRP6 study, and the same FZD2 fact, the In in GPC4. and made Frizzled that observation, Wnt3a to possibility bind for the simultaneously exclude Frizzled and can not Wnt3a because with does activity result compete signaling this Wnt to cannot However, of the binding. GPC4 more stimulation that a this where binding found excluded in they study GPC4 surface, this for by of that role cell authors direct proposed The the signaling located. 2012) are at al., receptors Wnt et Wnt (Sakane concentrating stimulate al. et glypicans Sakane FZD2, and the interact mediate chains Frizzled. GAG chains of these CRD GAG that the the found with we that addition, indicating In assay, interaction. proteins same Frizzled the the of any in to that bind showed pull-down cannot also GPC3 we and non-glycanated Notably, a FZD8. cells, and FZD7 293T FZD4, with transfected assays transiently in performed Frizzled-4. with interact (FZD FZD4 not tagged does GPC6 6. Fig. hi;H,hmgltnnA h oiin fmlclrms akr r niae ntelf.()Cl idn sa.23 el htwr transfect were that cells 8 293T at assay. h binding 2 Cell l for (B) immunoglobulin alone AP left. 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(A) ih o eal osiuaeWtatvt.Hwvr ehave we However, activity. Wnt stimulate to able GPC3 be non-glycanated not a that might suggests Frizzled and shown). non-canonical GPC3 not between inhibits (data also system this cellular GPC6 in same that the described in find signaling signaling Wnt we canonical because inhibitory the on study, explain GPC6 could that of rafts unlikely lipid activity it the of consider outside stimulate we localization to However, and signaling signaling. Wnt of canonical non-canonical outside inhibit 2012). localize to al., to only acts forced et rafts is signaling (Sakane these that rafts GPC4 Wnt these mutated of a non-canonical outside Significantly, is rafts, of GPC4 lipid stimulation when at occurs localized the be that Wnt- to has and the GPC4 to signaling, Wnt contribute canonical also could glypican. surface this levels is cell of the It activity increase the signaling. inhibitory to at GPC6 Wnt of Dkk1 canonical ability of of the that inhibitor therefore secreted possible provides a non- Dkk1, Wnt3a, to a to the with Knypek, bind that observed to not conclusion. cannot this is to support that effect additional level, inhibitory GPC6 signal-reception the the glycanated at that occurs signaling inhibition and GPC6-induced Wnt a the canonical as that of acts fact a therefore The and form inhibitor. 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Journal of Cell Science P3idcdsiuaino aoia n ciiymight activity Wnt canonical the in of chains GAG the stimulation on of Based role 2005). the GPC3-induced that al., speculated et we (Capurro results, PLC-PRF-5 these line for cell the HCC required the in of were signaling HLF chains Wnt3a line cell canonical GAG HCC of the the on stimulation example, GPC3- depends GPC3-induced the For signaling in context. Wnt chains GAG canonical cell the of of stimulation role induced the that previously shown ARTICLE RESEARCH Dp,oeo h two the of one (Dlp), in systems cell investigated. the been in have least at they to pathways, which regard signaling with Hh Hh and functions for Wnt opposing receptor the displays this GPC3 with interact Thus, not competes the does binding. therefore to but it Hh due and to likely al., Patched, affinity high most with et with is (Capurro binds GPC3 GPC3 embryo that of fact mouse activity the inhibitory in The 2008). signaling Hh of regulator is heparin by core 2011). replaced al., protein be et (Hh) not the (Li could Hedgehog alone activity that of al., this stimulation however, et because GPC5-induced signaling, (Li noted, the Patched be for receptor necessary should signaling It the Sonic 2011). ligand and chains for the this sulfate both (Shh) with heparan In glypican described hedgehog the this 2011). Notably, that of shown al., interaction that have the et mediate we chains. (Li GPC5, to of signaling case sulfate Hh different the of heparan stimulation significantly GPC5-induced the is the and protein, mechanism core through Frizzled, the Frizzled through of and mainly latter levels former Wnt the low between engaging 2009). interaction with by we the al., contexts finding, facilitate this might et cellular on GPC3 Wnts in Yan Based Frizzled. that, with 2005; for and propose essential GPC3 al., interaction are of chains et GAG interaction their the the Song that for show we 2005; here, required Interestingly, al., not et are (Capurro with Dally interaction and productive a with form in interaction cannot that, propose its Frizzled. Wnt to us leads that context, chains GAG observation this the by dimensionality al., our mediated the is et reduce diffusion, Wnt3a to (Schlessinger ligand able Frizzled also of is and GPC6 the Wnt Although reducing 1995). frequency between and the surface encounters increasing cell thus of the with diffusion, at Wnt ligand Wnt of of dimensionality to interaction non-glycanated binding productive Frizzled, by a of Frizzled facilitate levels still possible high could is it expressing GPC3 2005), al., cells directly et in (Capurro interact GPC3 can that, cell of Wnts the particular protein because core on a fact, the and/or by In with expressed 2005). involved, al., GPC3 Frizzled et and or (Capurro Frizzled Wnt Wnt, of of type levels the on depend eueteWtsiuaoyatvt.Teeae oee,two however, are, There to activity. manner Wnt-stimulatory competitive-inhibitory the surface, a the cell in the reduce act at Frizzled could expected than and glypican be glypican excess would Wnts more is it with there model, if complex this glypicans that to a that according forming Wnt-stimulatory proposing because, by Frizzled, model the signaling our These Wnt in shown). with stimulate not consistent reduction (data GPC3 are a of results concentrations observed high at at reduced. also activity is that stimulation have but Dlp-induced ratios, We the Dlp:Frizzled Dlp ratios low that Dlp:Frizzled showed at high authors signaling the Wnt cells, stimulates cultured in transfected signaling using Wnt By on activity regulatory thsbe eotdpeiul htteGGcan fGPC3 of chains GAG the that previously reported been has It eety a ta.(a ta. 09 eotdta Dally-like that reported 2009) al., et (Yan al. et Yan Recently, negative a as acts GPC3 that reported recently have We nvitro in Drosophila u hywr o eurdi h case the in required not were they but , lpcn,dsly biphasic a displays glypicans, Drosophila igdisks. wing 0 ea oiesrm(B) xrsinvcosfrGPC3, for vectors Expression (FBS). ATCC serum the from bovine obtained with supplemented GPC3 were DMEM fetal in L-Wnt3a grown were and 10% lines cell L All 293T, VA). (Manassas, lines cell transfections The and plasmids lines, Cell METHODS AND MATERIALS aei-epniepooe (TOPFLASH)], a a by with driven promoter co-transfected is expression were luciferase catenin-responsive and which plates [in vector 24-well reporter in luciferase seeded were cells 293T (sc- assay Biotechnology Luciferase P-LRP6 Cruz Wnt3a- Santa against from CA). and the Cruz, antibody was and Santa L- LRP6 CA), 25317, The (Danvers, total of Technology against Signaling added. of dilutions antibody Cell levels from other were was the (Ser1490) and indicated, medium lysed, When then conditioned western were by analysis. assessed Cells were blot LRP6 overnight h. total and 1 an (P-LRP6) LRP6 for After phosphorylated added serum-free FBS. with was 1:3 1% diluted medium medium containing Wnt3a-conditioned or medium was L- medium incubation, fresh the day, with expression following The GPC6 replaced vectors. or control GPC3 respective a or with vector, transfected transiently were cells 293T phosphorylation LRP6 of Assessment GPC6 of mutant non-glycanated A MA). (Harvard Toker Boston, Alex (GPC6 from School, obtained was Johns plasmid Medical DNA (The pCDNA GPC6 the Nathans human in Jeremy full-length inserted The from MD). Myc- gift Baltimore, Germany). a University, Hopkins Heidelberg, were (DKFZ, vectors Niehrs FZD-CRD Liliana tagged Christof by vector by provided Frizzled-8–YFP provided the were and was Canada), vectors Toronto, Frizzled of (University Gonzalez FLAG-tagged Attisano 2008; al., 1998). et al., (Capurro et previously described been have plasmid, N eunig P6A a eeae yisrigtehuman the inserting by generated the by into was cDNA verified GPC6 GPC6–AP were Mutations sequencing. mutagenesis. DNA site-directed by respectively, Ser opt ihFize o n idn,tu ciga ninhibitor an as will signaling. acting GPC3 Wnt thus secreted binding, of Wnt a for that the Frizzled expected at with is Frizzled compete 2005; and it secreted Wnt Conversely, al., a of surface. that interaction cell the expect et facilitate to not reasonable (Capurro will is consistent GPC3 It Wnt are lines report. here previous canonical presented our cell results inhibit with The 2010). 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Staccini-Lavenant, A., Gallet, L., K. Ayers, ihH-agdGC rGPC6 or GPC6 HA-tagged with uhrcontributions Author interests. competing no declare authors The interests Competing were cells transfection, after day 1 At control. or a FZD8 8 to FZD7, as transferred FZD4, FLAG-tagged pCDNA with with transfected were cells 293T assay Cell-binding aeao . un,Y . tut . aa . hed,R,Kazanskaya, R., Ahrendt, M., Tada, N., Staudt, L., Y. Huang, L., Caneparo, H. Nakato, and O. Shimmi, S., Takeo, C., Firkus, K., Kamimura, T., Research. Akiyama, Health of Institute References Canadian the by funded been has work This Funding the wrote J.F. and M.C. experiments, the manuscript. performed T.M. and W.S. M.C., h ellrpopaae,adteA ciiywste esrdwt a with measured then was activity AP fast the Sigma and phosphatases, cellular the iheulaon fpoen eehae o65 aliquots cells to Lysate the heated NP40. were 1% PBS, proteins containing of 8 with amount pH equal washes Tris-HCl with mM four h. 10 by 2 in for removed lysed cells were the was to ligand added were unbound activity After AP of amount same the containing ewe h niae rtiswsqatfe yuigPearson’s using twice. Inc., immunostaining performed from by caveolin was cells the experiment Zeiss Only representative quantified experiments. 14 independent three (Carl of was least minimum at a SP2 proteins using Colocalization coefficient, a indicated Browser. v3.2 correlation Image the using LSM 510 Zeiss between generated and LSM Canada) were ON, microscope 12CA5 anti- Pickering, Images laser anti-HA donkey Confocal scanning mouse Alexa-Fluor-488-conjugated (Invitrogen). with and For mouse-IgG detected medium. antibody Wnt3a-conditioned was monoclonal or GPC6 L- with immunostaining, incubated and coverslips, lvr,H. Clevers, J. Filmus, and F. Li, I., M. Capurro, Iozzo, and T. Neill, T., R. Owens, N., Tyler-Rubinstein, N., Pal, S., Buraschi, R. Nusse, and T. J. Blitzer, M. Bienz, M., C. Cruciat, T., Zimmermann, G., Davidson, L., Y. Huang, J., Bilic, Lefranc M., M. Souza, de A., Bengochea, Lin, and H. Liu, M., Khodoun, J., R. Opoka, D., Yan, C., Han, Y., T. Belenkaya, aur,M . u . h,W,L,F,Ja .adFlu,J. 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